Research is proposed to develop a novel mammalian cell mutagenesis assay in which mutation or loss of the p53 tumor suppressor gene can be efficiently and economically assessed using L5178y/tk+/-3.7.2C cells, which were recently found to be heterozygous for p53 as well as for tk. An alternate hypothesis for the bimodal distribution of mutant colony sizes observed in the mouse lymphoma assay (MLA) suggests that large lambda colony mutants result from mutation or loss of one allele, tk+, and that small sigma colony mutants result from mutation or loss of two alleles, tk+ and p53-, both found on the distal 15% of chromosome 11b In 3.7.2C cells; thus, the growth rates of cells mutated at the tk locus depend upon whether the dominant negative p53 genotype is retained or lost. Recovery of sigma colony mutants is greatly enhanced In an in situ MLA (ISMLA) in which cells are cloned after chemical exposure, and preliminary results suggest that even greater precision may be obtained if the cells are cloned in microtiter (MT) plates instead of in soft agar. In Phase I, an economical multi-locus (ML) MT-ISMLA will be defined in which the p53 phenotype of lambda and sigma colony mutants will be assessed by immunohistochemical staining; it will be automated and validated in Phase II. An accurate and economical MLMT ISMLA which assesses mutations and loss of heterozygosity at p53 as well as tk will be significantly more relevant than conventional MLA protocols and numerous other short-term tests for examining mechanisms of mutagenesis and clastogenesis, predicting carcinogenesis, evaluating chemicals for regulatory submissions, and risk assessment. PROPOSED COMMERCIAL APPLICATION: The ML-MT ISMLA should be in high demand after it has been precisely defined, automated, validated, and accepted by regulatory agencies. Potential commercial applications include marketing MT-ML ISMLA research and testing to the government and industry, holding training workshops, offering MLA colony p53 immunohistochemical staining services, consulting services related to use of the assay and interpretation of its results, and commercialization of an image analysis system for this assay.